This project focuses on the expression, immunological properties, and structure-function relationships of peptide hormone receptors. (1) We expressed the full-length and 1-240 residue segment of the rat angiotensin II (Ang II) type 1b receptor (AT1b) in baculovirus/Sf9 insect cell systems as non-fused protein and glutathione-S-transferase fusion proteins. Western blot analysis using a AT1 specific antiserum raised against the C-terminal segment (LE92, residues 268-369) of the AT1b receptor revealed bands ranging from 66-210 kDa. The recombinant proteins were found mostly in the inclusion bodies and none was detected in the soluble fraction or medum, even when a conventional secretory signal sequence was included in the vector. Ang II binding activity was detectable but was lower than that of partially purified bovine adrenal membranes (BAM). (2) The human AT1 receptor was expressed using full length cDNA from a plasmid vector containing a 6XHis tag, and an baculovirus vector/cabbage looper cell system. Ang II binding analysis of the optimally infected cells showed 90 times more binding sites than those found in the BAM on a protein weight basis. However, the majority of the receptor protein was found in the inclusion bodies, with bands in the 66-210 kDa range as revealed by Western blot analysis using anti-LE92 serum. It is probable that the receptors expressed on the plasma membrane are those that display hormone-binding activity. Purification of the receptor protein by Ni-affinity chromatography is in progress. (3) In addition to seven chemically synthesized segments of the GnRH receptor, three long segments (residues 19-77, 138-194, and 265-359) each fused with maltose-binding protein were expressed in E. coli. Milligram quantities of the fusion proteins were obtained after maltose-affinity chromatography, and were used to generate antibodies in rabbits. After Factor Xa cleavage, the fragments yielded N-terminal amino acid sequences consistent with those of the respective receptor fragments.